Abstract Glycans linked to Asparagine (Asn) (N-glycans, N-glycome) and Serine/Threonine (Ser/Thr) (O-glycans, O- glycome), play critical roles in the molecular interactions that govern many important biological systems. Furthermore, alterations in these glycomes, often associated with pathological states, and can significantly affect cellular properties and functions. In order to fully understand the roles of glycans in biology, functional glycomics, information on specific cellular glycomes is necessary. Glycan analysis, however, often requires release of glycoconjugates prior to structural analysis by modern technologies, such as Mass Spectrometry (MS). While N-glycan analyses are facilitated by the availability of endo-glycosidases that affect their release, no such comparable method exists for O-glycans. Instead, release of O-glycans from glycoproteins often relies on ?-elimination, which has many limitations, such as non-specificity, and low efficiency, and the requirement for large amounts of material. The technology for quantitative analysis of glycans, also known as quantitive glycomics, is even more limited. Similarly, the preparation of complex glycans for biological studies is extremely challenging, since only limited numbers and amounts of glycans can be chemically or chemoenzymatically synthesized or isolated from natural sources. To overcome these issues, we are developing a novel technology termed Cell O-glycome Reporter/Amplification(CORA)- a simple, sensitive, and versatile method for profiling mucin type O-glycan structures and amplifying/preparing complex O-glycans from cultured cells fed with peracetylated Benzyl-?-N-acetylgalactosamine [Bn-?-GalNAc(AC)3]. The products, Bn-?- O-Glycans synthesized by cells are secreted into the media and easily isolated for analysis by MS. Our CORA technology has demonstrated high sensitivity, reproducibility, accuracy, and feasibility as well as amplification nature. In this application, we propose to advance our current CORA technology into the Quantitative-CORA and Preparative-CORA for quantitative O-glycomics and amplification/preparation of cellular O-glycans by a study through 4 following Aims. Aim 1: To establish and validate Quantitative-CORA by developing ?metabolic Stable Isotopic Labeling of O-glycomes of cultured Cells (SILOC)? with [13C6]Bn-?-GalNAc(Ac)3 for comparative quantitive O-glycomics; Aim 2: To develop Preparative-CORA using 2-PA-5-O-?-GalNAc(Ac)3 which contains a fluorescent tag posing an active Amine group for immobilizing; Aim 3: To validate the Preparative-CORA technology by amplifying and preparing cellular O-glycomes for 20 cell lines using cells as ?O-Glycan factories?; Aim 4: To prepare individually purified complex O-glycans from amplified cellular O-glycomes for biological function studies by combining Preparative-CORA, 2D-HPLC Separation and Glycan Microarray technologies. These Quantitative-CORA and Preparative-CORA technologies will meet the critical demands in the field and directly facilitate both cellular O-glycomics and functional glycomics studies.